Esophageal Cancer Tissues Research Articles

FAT1 and PTPN14 Regulate the Malignant Progression and Chemotherapy Resistance of Esophageal Cancer through the Hippo Signaling Pathway.

Background Esophageal cancer (EC) is a common malignant tumor, which brings heavy economic burden to patients and society. Therefore, it is important to understand the molecular mechanism of recurrence, metastasis, and drug resistance of esophageal cancer. Methods Human esophageal cancer cell line TE13 (poorly differentiated squamous cell carcinoma) and normal human esophageal epithelial cell line het-1a were selected for aseptic culture. At the same time, 6 bottles of TE13 cell line were inoculated in logarithmic phase. Cell apoptosis was analyzed by flow cytometry (FCM). Cell clone formation assay was used to analyze the proliferation. Fibronectin-coated dishes were used to detect the characteristics of cell adhesion to extracellular matrix. The Transwell method was used to detect the cell invasion ability. Western blot was used to analyze the expression of Yap1, PTPN14, FAT1, and Myc. Results Results showed that FAT1 and PTPN14 were downregulated, while Yap1 was upregulated in esophageal cancer tissues. FAT1 inhibited the proliferation, adhesion, and invasion of human esophageal cancer cell lines, which might be associated with the upregulation of PTPN14 and the inhibition of Yap1 and Myc. Conclusion The results suggested that PTPN14 and FAT1 could regulate malignant progression and chemotherapy resistance of esophageal cancer based on the Hippo signaling pathway.

Targeting lectin-like oxidized low-density lipoprotein receptor-1 triggers autophagic program in esophageal cancer.

Autophagy is a highly conserved catabolic process to maintain cellular homeostasis. However, dysfunctional autophagy contributes to a context-dependent role in cancer. Here, we clarified the exact role of autophagy modulated by the scavenger receptor lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in esophageal cancer (EC). A comprehensive analysis in various cancers displayed that LOX-1 was upregulated the most in EC tissues and associated with poor prognosis of patients. Deletion of LOX-1 ex vivo and in vivo suppresses EC development by inducing autophagic cell death. Receptor for activated C kinase 1 (RACK1) was identified as a signal adapter of LOX-1, which incented RAS/MEK/ERK pathway and TFEB nuclear export signal and safeguarded tumorigenesis. A sulfated polysaccharide fucoidan extracted from brown seaweed was found to bind with LOX-1 and mediate its proteasomal degradation but not the lysosome pathway, leading to autophagy-related cell death in EC. These results reveal a central contribution of LOX-1 to EC development and provide genetic ablation or bioactive polysaccharide as an effective intervention for EC therapy.

Long non-coding RNA HAGLROS regulates the proliferation, migration, and apoptosis of esophageal cancer cells via the HAGLROS-miR-206-NOTCH3 axis.

Esophageal cancer (EC) is a common malignant tumor of the digestive tract, the treatment of which involves surgery combined with radiotherapy and chemotherapy, as well as other comprehensive types of treatment. The pathogenesis of EC remains unclear, which hinders the development of clinical therapy and the identification of molecular targets for this disease. Long non-coding RNAs (lncRNAs) have been shown to be associated with the malignant biological behavior of EC, but the specific molecular mechanisms underlying the carcinogenesis of EC are not fully understood. Reverse transcription-quantitative PCR (RT-qPCR) was applied to measure the lncRNA HAGLR opposite strand lncRNA (HAGLROS) levels in EC cell lines and tissues. Cell Counting Kit-8 (CCK-8) detection, scratch test, and Transwell assay were performed to determine the proliferation, migration and invasion of EC cell. The interaction between HAGLROS, microRNA (miR)-206, and notch receptor 3 (NOTCH3) was confirmed by RNA immunoprecipitation and dual luciferase reporter gene assays. HAGLROS is upregulated in esophageal squamous cell carcinoma (ESCC) tissues and predicts poor prognosis. Silent HAGLROS is negatively associated with malignant behavior in EC cells. Low expression of HAGLROS can induce decreased invasive and migratory abilities in EC cells. Downregulated HAGLROS significantly inhibits the proliferation of EC cells and accelerates apoptosis. HAGLROS promotes EC cell tumorigenesis in vivo. HAGLROS participates in the HAGLROS/miR-206/NOTCH3 regulatory axis in EC cells. HAGLROS may play a role in the progression of EC by modulating the miR-206/NOTCH3 signaling axis, and may be a novel target for the diagnosis and treatment of EC.

Expression and function analysis of CRABP2 and FABP5, and their ratio in esophageal squamous cell carcinoma.

ObjectiveThe purpose of this study was to explore the effect of CRABP2 and FABP5, and their ratio on prognosis in esophageal squamous cell carcinoma.MethodsThe expression data of CRABP2 in esophageal cancer in TCGA and GEO were collected by the public database GEPIA. The expression levels of CRABP2 and FABP5 were examined using immunohistochemistry. The relationship between the two proteins and related clinicopathological parameters were analyzed by χ 2 test. Survival analysis was used to investigate the effect of CRABP2 and FABP5, and their ratio on prognosis.ResultsCompared with normal esophageal mucosal epithelium, there was lower CRABP2 gene mRNA in the esophageal cancer tissue, and the difference was statistically significant (p < 0.01). For the expression level, no significant difference was observed in patients with stages I–IV in esophageal cancer. Immunohistochemistry showed that CRABP2 and FABP5 were both highly expressed in normal esophageal squamous epithelial cells at 100 and 94.1%, while lower in ESCC (75.6 and 58.7%). There was a significant difference in the expression between cancer and adjacent tissues (p < 0.001). No inherent relationship was manifested between the CRABP2 expression and the clinical parameters of the ESCC. The expression of FABP5 was related to lymph node metastasis (p = 0.032), the depth of invasion (p = 0.041), and the AJCC stage (p = 0.013). The ratio of CRABP2 and FABP5 was related to ethnicity (p = 0.001), nerve invasion (p = 0.031), and postoperative treatment (p = 0.038). CRABP2 is positively associated with FABP5 (r = 0.156, p = 0.041) and the ratio (r = 0.334, p = 0.000), while there was a negative correlation between FABP5 and the ratio (r = −0.269, p = 0.000). Patients with CRABP2-positive expression had a significantly longer overall survival than patients with CRABP2-negative expression (p = 0.025).ConclusionCRABP2 as a suppressor factor is expected to be a potential prognosis marker for esophageal squamous cell carcinoma.

LncRNA RP11-465B22.8 triggers esophageal cancer progression by targeting miR-765/KLK4 axis

LncRNAs play an important role in tumorigenesis and progression; however, the function and mechanisms of lncRNAs in esophageal cancer (EC) remain largely unclear. In this study, we screened the differentially expressed lncRNAs in EC by using RNA-seq and one of the most upregulated lncRNAs, lncRNA RP11-465B22.8, was further characterized. LncRNA RP11-465B22.8 was upregulated in EC tissues and high lncRNA RP11-465B22.8 expression was associated with poor survival of EC patients. Ectopic expression of lncRNA RP11-465B22.8 enhanced the proliferation, migration, and invasion of EC cells, whereas knockdown of lncRNA RP11-465B22.8 led to the opposite effects. Mechanistically, lncRNA RP11-465B22.8 sponged miR-765 to increase the expression of KLK4. Moreover, LncRNA RP11-465B22.8 could be delivered from EC cells to macrophages via exosomes and subsequently induced M2 macrophage-induced cell migration and invasion. Our findings revealed a novel lncRNA RP11-465B22.8/miR-765/KLK4 pathway in EC and indicated that lncRNA RP11-465B22.8 might be a potential target for EC therapy.

Therapeutic Potential of the Cyclin-Dependent Kinase Inhibitor Flavopiridol on c-Myc Overexpressing Esophageal Cancer.

Tumors with elevated c-Myc expression often exhibit a highly aggressive phenotype, and c-Myc amplification has been shown to be frequent in esophageal cancer. Emerging data suggests that synthetic lethal interactions between c-Myc pathway activation and small molecules inhibition involved in cell cycle signaling can be therapeutically exploited to preferentially kill tumor cells. We therefore investigated whether exploiting elevated c-Myc expression is effective in treating esophageal cancer with the CDK inhibitor flavopiridol. We found frequent overexpression of c-Myc in human esophageal cancer cell lines and tissues. c-Myc overexpression correlated with accelerated esophageal cancer subcutaneous xenograft tumor growth. Esophageal cancer cells with elevated c-Myc expression were found preferentially more sensitive to induction of apoptosis by the CDK inhibition flavopiridol compared to esophageal cancer cells with lower c-Myc expression. In addition, we observed that flavopiridol alone or in combination with the chemotherapeutic agent nanoparticle albumin-bound paclitaxel (NPT) or in combinations with the targeted agent BMS-754807 significantly inhibited esophageal cancer cell proliferation and subcutaneous xenograft tumor growth while significantly enhancing overall mice survival. These results indicate that aggressive esophageal cancer cells with elevated c-Myc expression are sensitive to the CDK inhibitor flavopiridol, and that flavopiridol alone or in combination can be a potential therapy for c-Myc overexpressing esophageal cancer.

Metabolomic Characterization Reveals ILF2 and ILF3 Affected Metabolic Adaptions in Esophageal Squamous Cell Carcinoma.

Esophageal cancer (EC) is a common malignant disease in eastern countries. However, a study of the metabolomic characteristics associated with other biological factors in esophageal squamous cell carcinoma (ESCC) is limited. Interleukin enhancer binding factor 2 (ILF2) and ILF3, double-stranded RNA-binding proteins, have been reported to contribute to the occurrence and development of various types of malignancy. Nevertheless, the underlying functions of ILF2 and ILF3 in ESCC metabolic reprogramming have never been reported. This study aimed to contribute to the metabolic characterization of ESCC and to investigate the metabolomic alterations associated with ILF2 and ILF3 in ESCC tissues. Here, we identified 112 differential metabolites, which were mainly enriched in phosphatidylcholine biosynthesis, fatty acid metabolism, and amino acid metabolism pathways, based on liquid chromatography–mass spectrometry and capillary electrophoresis–mass spectrometry approaches using ESCC tissues and paired para-cancer tissues from twenty-eight ESCC patients. In addition, ILF2 and ILF3 expression were significantly elevated in EC tissues compared to the histologically normal samples, and closely associated with PI3K/AKT and MAPK signaling pathways in ESCC. Moreover, in ESCC tissues with a high ILF2 expression, several short-chain acyl-carnitines (C3:0, C4:0, and C5:0) related to the BCAA metabolic pathway and long-chain acyl-carnitines (C14:0, C16:0, C16:0-OH, and C18:0) involved in the oxidation of fatty acids were obviously upregulated. Additionally, a series of intermediate metabolites involved in the glycolysis pathway, including G6P/F6P, F1,6BP, DHAP, G3P, and 2,3BPG, were remarkably downregulated in highly ILF3-expressed ESCC tissues compared with the corresponding para-cancer tissues. Overall, these findings may provide evidence for the roles of ILF2 and ILF3 during the process of ESCC metabolic alterations, and new insights into the development of early diagnosis and treatment for ESCC. Further investigation is needed to clarify the underlying mechanism of ILF2 and ILF3 on acyl-carnitines and the glycolysis pathway, respectively.

Sesamin exerts anti‐tumor activity in esophageal squamous cell carcinoma via inhibition of TRIM44 and NF‐κB signaling

Tripartite motif-containing 44 (TRIM44) is known to play an oncogenic role in multiple human cancers, including esophageal cancer. Sesamin possesses potent anti-inflammatory and anti-cancer properties for various cancers. This study is designed to unravel the biological functions of sesamin and TRIM44 in esophageal cancer. TRIM44 expression in esophageal squamous cell cancer (ESCC) cell lines and tissues was determined by RT-qPCR assay and Western blot. The effects of sesamin and TRIM44 on ESCC cell growth in vivo and in vitro were assessed by the mouse model and CCK-8 assay, respectively. We found that TRIM44 was significantly upregulated in ESCC cell lines and tissues when compared to their counterparts. Sesamin treatment or depletion of TRIM44 markedly reduced ESCC cell proliferation. The nuclear factor kappa B (NF-κB) and toll-like receptor 4 (TLR4) signaling pathway may be involved in sesamin-mediated TRIM44 suppression. Finally, we showed that oral administration of sesamin dramatically inhibited tumor growth or ESCC in nude mice. Our results suggest that sesamin exerts anti-tumor activity in ESCC via inhibition of NF-κB signaling pathway, demonstrating its potential for the treatment of esophageal cancer.

Association of Serum Anti-PCSK9 Antibody Levels with Favorable Postoperative Prognosis in Esophageal Cancer.

BackgroundEsophageal cancer often appears as postoperative metastasis or recurrence after radical surgery. Although we had previously reported that serum programmed cell death ligand 1 (PD-L1) level correlated with the prognosis of esophageal cancer, further novel biomarkers are required for more precise prediction of the prognosis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with the cholesterol metabolism. But there was no report of relationship between serum PCSK9 antibody and cancer. Therefore, we investigated whether anti-PCSK9 antibodies could be a novel biomarker for solid cancer.MethodsSerum levels of anti-PCSK9 antibodies and antigens in patients with solid cancer were analyzed using amplified luminescence proximity homogeneous assay-linked immunosorbent assay (AlphaLISA). The reactivity of serum antibodies against recombinant PCSK9 protein was investigated by Western blotting, and the expression of PCSK9 antigens in esophageal cancer tissues was examined by immunohistochemical staining.ResultsAlphaLISA showed that serum anti-PCSK9 antibody (s-PCSK9-Ab) levels were significantly higher in patients with esophageal cancer, gastric cancer, colorectal cancer, lung cancer, and breast cancer than in healthy donors, and patients with esophageal cancer had the highest levels. The presence of serum antibody in patients was confirmed by Western blotting. There was no apparent correlation between s-PCSK9-Ab and PCSK9 antigen levels. Immunohistochemical staining demonstrated the expression of PCSK9 antigen in both the cytoplasm and nuclear compartments of esophageal squamous cell carcinoma tissue but not in normal tissue. Compared with patients with low s-PCSK9-Ab levels, those with high s-PCSK9-Ab levels had a favorable postoperative prognosis after radical surgery for esophageal cancer. In the multivariate analysis, tumor depth and s-PCSK9-Ab level were identified as independent prognostic factors. In the univariate analysis of clinicopathological features, high PCSK9 antibody levels were not associated with sex, age, location, tumor depth, lymph node status, squamous cell carcinoma antigen, or p53-Ab, whereas they correlated significantly with PD-L1 levels, which were associated with unfavorable prognosis. Correlation between s-PCSK9-Ab and PD-L1 levels was also confirmed in the logistic regression analysis; therefore, low s-PCSK9-Ab levels could discriminate another poor prognosis group other than high-PD-L1 group.ConclusionsPatients with solid cancer had higher s-PCSK9-Ab levels than healthy donors. High s-PCSK9-Ab levels indicated better prognosis for overall survival after surgery in patients with esophageal cancer.

Network analysis of miRNA targeting m6A-related genes in patients with esophageal cancer.

BackgroundWe investigated the miRNA-m6A related gene network and identified a miRNA-based prognostic signature in patients with esophageal cancer using integrated genomic analysis.MethodsWe obtained expression data for m6A-related genes and miRNAs from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. Survival analysis was conducted to identify potential prognostic biomarkers. LASSO Cox regression was performed to construct the overall survival (OS) associated prediction signature. We used the Kaplan–Meier (K–M) curve and receiver operating characteristic (ROC) curves to explore the signature’s efficiency and accuracy. Interactions between the m6A-related genes and miRNAs were identified in starBase3.0 and used to construct the miRNA-m6A related gene network.ResultsWe found that HNRNPC, YTHDF, ZC3H13, YTHDC2, and METTL14 were dysregulated in esophageal cancer tissues. Multivariate Cox regression analysis revealed that HNRNPC may be an independent risk factor for OS. Five hundred twenty-two potential upstream miRNAs were obtained from starBase3.0. Four miRNAs (miR-186, miR-320c, miR-320d, and miR-320b) were used to construct a prognostic signature, which could serve as a prognostic predictor independent from routine clinicopathological features. Finally, we constructed a key miRNA-m6A related gene network and used one m6A-related gene and four miRNAs associated with the prognosis. The results of our bioinformatics analysis were successfully validated in the human esophageal carcinoma cell lines KYSE30 and TE-1.ConclusionOur study identified a 4-miRNA prognostic signature and established a key miRNA-m6A related gene network. These tools may reliably assist with esophageal cancer patient prognosis.

Mitochondrial calcium uniporter promotes cell proliferation and migration in esophageal cancer.

Increasing evidence has suggested that mitochondrial calcium uniporter (MCU) is involved in various types of cancer. However, its functions remain unclear in esophageal cancer. The aim of the present study was to explore its abnormal expression and clinical implications in esophageal cancer. A total of 110 patients with esophageal cancer were enrolled in the study. Western blotting was performed to examine the protein expression levels of MCU in 8 pairs of esophageal cancer and adjacent normal tissues. Using immunochemistry, a total of 110 esophageal cancer specimens were analyzed to identify the association between MCU expression and clinicopathological features of patients with esophageal cancer. Furthermore, immunofluorescence of MCU was performed. Pearson's correlation analysis was performed between MCU and hypoxia inducible factor (HIF)-1α/VEGF/E-cadherin/Vimentin expression based on western blotting. After KYSE-150 and TE-1 cells were treated with the MCU agonist Spermine and a small interfering RNA against MCU (si-MCU), a series of functional assays were performed, including Cell Counting Kit-8, colony formation and Transwell assays. The results revealed that, compared with in adjacent normal tissues, MCU was highly expressed in esophageal cancer tissues. MCU expression was significantly associated with depth of invasion, lymph node metastasis, TNM stage and distant metastasis. Moreover, MCU was significantly correlated with HIF-1α/VEGF/E-cadherin/Vimentin in esophageal cancer tissues. MCU overexpression promoted VEGF, MMP2, Vimentin and N-cadherin expression, while it inhibited E-cadherin expression in KYSE-150 and TE-1 cells, and opposite results were observed after transfection with si-MCU. Furthermore, MCU overexpression accelerated the proliferation and migration of KYSE-150 and TE-1 cells. Thus, the current findings suggested that high MCU expression may participate in cell proliferation, migration and epithelial-mesenchymal transition in esophageal cancer.

LncRNA ASB16-AS1 regulates the proliferation, migration and invasion of esophageal cancer cells by targeting miR-1258

Objective: To investigate the effects of long-chain non-coding RNA ASB16 antisense RNA1 (ASB16-AS1) on the proliferation, migration and invasion of esophageal cancer cells by targeting microRNA (miR )-1258. Methods: Forty pairs of esophageal cancer tissues and matched adjacent tissues (distance of tumor margin>3 cm) resected in Xinxiang Central Hospital from May 2016 to July 2017 were collected. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expressions of ASB16-AS1 and miR-1258 in esophageal cancer tissues and adjacent tissues. The small interfering RNA negative control (si-NC), ASB16-AS1 small interfering RNA (si-ASB16-AS1), miR-negative control mimics (miR-NC), miR-1258 mimics (miR-1258), si-ASB16-AS1 and anti-miR-NC, si-ASB16-AS1 and anti-miR-1258, si-ASB16-AS1 and anti-miR-1258 were transfected into Eca109 cells, respectively. Methyl thiazolyl tetrazolium (MTT) was utilized to detect the cell viability. Transwell assays were applied to detect cell migration and invasion. Double luciferase reporting experiment and qRT-PCR were used to confirm the relationship between ASB16-AS1 and miR-1258. Results: The expression levels of ASB16-AS1 and miR-1258 in esophageal cancer tissues were 2.95±0.27 and 0.62±0.06, respectively. Compared with 1.00±0.06 and 1.00±0.07 in adjacent tissues, the difference was statistically significant (P<0.05). The cell viability of the si-NC group at 48 h and 72 h were 0.81±0.07 and 1.15±0.11, while those of si-ASB16-AS1 group were 0.46±0.04 and 0.62±0.06 (P<0.05). The numbers of cell migration and invasion in the si-NC group were 86.32±8.24 and 71.29±7.15, respectively, while those of si-ASB16-AS1 group were 43.22±4.31 and 32.36±3.58, respectively, the differences were statistically significant (P<0.05). The cell viability of the miR-NC group at 48 h and 72 h were 0.84±0.08, 1.18±0.12, while those of miR-1258 group were 0.55±0.05, 0.71±0.07 (P<0.05). The migration and invasion numbers of the miR-NC group were (83.15±8.31) and (75.33±7.51), while those of miR-1258 group were (49.58±4.23) and (38.42±3.84), respectively, the differences were statistically significant (P<0.05). The cell viability of the si-ASB16-AS1+ anti-miR-NC group at 48 h and 72 h were 0.45±0.04, 0.61±0.06, while those of si-ASB16-AS1+ anti-miR-1258 group were 0.72±0.07, 0.98±0.08; The migration and invasion numbers of cells in the si-ASB16-AS1+ anti-miR-NC group were 44.36±4.41 and 31.69±3.85, respectively, while those of si-ASB16-AS1+ anti-miR-1258 group were 72.65±7.27 and 61.22±6.14, respectively, and the differences were statistically significant (P<0.05). ASB16-AS1 targeted negative regulation of miR-1258 expression. Conclusions: ASB16-AS1 upregulates in esophageal cancer. ASB16-AS1 promotes the proliferation, migration and invasion of esophageal cancer cells by targeting miR-1258.

Poly(A)-Specific Ribonuclease (PARN) Promotes the Proliferation, Migration and Metastasis of Esophageal Cancer

Background: Esophageal cancer leads to the majority of cancer deaths in the world. Surgery, radiotherapy and chemotherapy are the main treatment methods, but due to its high recurrence and metastasis rate, the overall prognosis is poor. Poly(A)-specific ribonuclease (PARN) is a multifunctional enzyme molecule, which is involved in the occurrence and development of tumors. This study explores the relationship between PARN and the proliferation, metastasis and prognosis of esophageal cancer. Methods: The esophageal cancer tissues and adjacent normal tissues were collected from 91 patients with esophageal cancer after surgery. The expression level of PARN was detected by immunohistochemistry (IHC) using tissue microarray (TMA). PARN expression and its relationships with clinicopathological characteristics and prognosis was detected. In addition, stable PARN knockdown esophageal cancer cell lines TE-1 and Eca-109 were used to study its effect on the phenotype and behavior of tumor cells. Meanwhile, the effect of PARN silencing on tumor growth in vivo was verified by xenotransplantation model in nude mice. Finally, we examined possible downstream signaling mechanisms of PARN in esophageal cancer. Findings: PARN showed high expression in esophageal carcinoma tissues and its expression was significantly correlated with lymphatic metastasis and prognosis of patients. In the culture of PARN knockdown esophageal cancer cell lines, the growth and proliferation were significantly inhibited, and the invasion and migration ability of tumor cells were also significantly decreased. In addition, inhibition of PARN suppressed tumor growth and increased survival time in xenograft murine models. In the PARN knockdown group, BIM, IGFBP-5 and p21 were significantly increased, while the expression levels of anti-apoptotic proteins Survivin and STNF-R1 were significantly down-regulated. In addition, the expression levels of Akt, p-Akt, PIK3CA and CCND1 downstream of the signaling pathway regulating esophageal cancer progression were significantly down-regulated. Interpretation: These results suggest a previously undetected role of PARN as a suppressor of tumor development and its associations of prognosis in esophageal cancer through anti-apoptotic effect. Funding Information: None. Declaration of Interests: The authors declare no conflicts of interests. Ethics Approval Statement: Ethical approval for the study was approved by the Research Ethics Committee of the Second Affiliated Hospital of Guangxi Medical University and Zhujiang Hospital Affiliated to Southern Medical University, and written patient consent was obtained from all patients. Nude mice xenograft model - This study was approved by the Guangxi Medical University Ethics Committee.

Long non-coding RNA OIP5-AS1 promotes the progression of esophageal cancer by regulating miR-30a/VOPP1 expression.

Long non-coding RNAs (lncRNAs) serve an important role in the development of esophageal cancer (EC), which is the eighth most common type of cancer worldwide. lncRNA opa-interacting protein 5 antisense transcript 1 (OIP5-AS1) is associated with human malignancy. However, the biological roles of OIP5-AS1 in the development of EC remain unclear. In the present study, transfection was conducted, and reverse transcription-quantitative PCR and western blot analysis were used for the detection of mRNA and protein expression, respectively. Furthermore, dual-luciferase reporter and RNA immunoprecipitation assays were used to study the interaction between miRNA and lncRNA or genes. The results revealed that OIP5-AS1 expression in EC tissues and cultured EC cells was upregulated, microRNA-30a (miR-30a) expression was downregulated. OIP5-AS1-knockdown suppressed the proliferation, migration and invasion of EC9706 and EC109 cells. miR-30a was confirmed to interact with OIP5-AS1, and miR-30a-mimics transfection ameliorated the effects of OIP5-AS1 in EC cells. Vesicular overexpressed in cancer prosurvival protein 1 (VOPP1) was verified as the direct target of miR-30a. VOPP1 expression was positively correlated with OIP5-AS1 expression in EC cells. Overexpression of VOPP1 ameliorated the negative effects of OIP5-AS1-knockdown on EC9706 and EC109 cells. In conclusion, OIP5-AS1 promoted the proliferation, migration and invasion of EC cells by increasing VOPP1 expression by sponging miR-30a.

Exosomes Mediated Transfer of Circ_0000337 Contributes to Cisplatin (CDDP) Resistance of Esophageal Cancer by Regulating JAK2 via miR-377-3p.

Background: Chemoresistance remains a major obstacle to the treatment of esophageal cancer patients. Exosome-mediated transfer of circular RNAs (circRNAs) has been reported to be related to drug resistance in esophageal cancer. This study is designed to explore the role and mechanism of exosomal circ_0000337 on CDDP resistance in esophageal cancer.Methods: Cell viability, proliferation, colony number, apoptosis, migration, and invasion were assessed by Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry, and transwell assays. Circ_0000337, microRNA-377 (miR-377-3p), and Janus kinase 2 (JAK2) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Exosomes were isolated and detected by differential centrifugation and a transmission electron microscope. Protein levels of CD9, CD63, and JAK2 were tested by Western blot assay. The binding relationship between miR-377-3p and circ_0000337 or JAK2 was predicted by circinteractome or Starbase and then verified by dual-luciferase reporter assay and RNA pull-down assay. The biological role of exosomal circ_0000337 and CDDP on esophageal cancer cell growth was examined by the xenograft tumor model in vivo.Results: Circ_0000337 and JAK2 were highly expressed, and miR-377-3p was decreased in CDDP-resistant esophageal cancer tissues and cells. Moreover, circ_0000337-containing exosomes secreted by CDDP-resistant esophageal cancer cells could promote CDDP resistance, cell growth, and metastasis in CDDP-sensitive esophageal cancer cells in vitro. The mechanical analysis discovered that circ_0000337 functioned as a sponge of miR-377-3p to regulate JAK2 expression. Exosomal circ_0000337 increased the drug resistance of esophageal cancer in vivo.Conclusion: Exosomal circ_0000337 accelerated CDDP resistance of esophageal cancer cells partly by regulating the miR-377-3p/JAK2 axis, hinting a promising therapeutic target for the esophageal cancer treatment.

Abstract 975: Antitumor effect of cyclin- dependent kinase inhibitor flavopiridol in c-Myc overexpressing esophageal cancer

Abstract Tumors with elevated c-Myc expression often exhibit highly aggressive phenotype and c-Myc amplification has been shown to be frequent in esophageal cancer. Emerging data suggests that synthetic lethal interactions between c-Myc pathway activation and small molecules inhibition involved in cell cycle signaling can be therapeutically exploited to preferentially kill tumor cells. We therefore investigated whether exploiting a synthetic-lethal approach dependent on elevated c-Myc signaling is effective in treating esophageal cancer with a CDK inhibitor flavopiridol. We found frequent overexpression of c-Myc in esophageal cancer cell lines and tissues. c-Myc overexpression correlated with accelerated esophageal cancer subcutaneous xenograft tumor growth without association with improved overall survival in esophageal cancer patients. c-Myc expression was enhanced by Wnt signaling activator (6-bromoindirubin-3′ -oxime (BIO) or pcDNA3-cMyc and c-Myc expression was depleted by c-Myc siRNAs. Interestingly, esophageal cancer cells with elevated c-Myc expression were preferentially more sensitive to induction of apoptosis by CDK inhibition flavopiridol compared to esophageal cancer cells with lower c-Myc expression. In addition, we observed that flavopiridol alone or in combinations with chemotherapeutic agent nanoparticle albumin-bound paclitaxel (NPT) or in combinations with targeted agent BMS-754807 significantly inhibited esophageal cancer cell proliferation and subcutaneous xenograft tumor growth with significantly enhancing overall mice survival. These results indicate that aggressive esophageal cancer cells with elevated c-Myc expression are sensitive to CDK inhibitor flavopiridol and flavopiridol alone or in combination with cytotoxic or targeted agents can be a potential therapy for c-Myc overexpressing esophageal cancer. Citation Format: Md Sazzad Hassan, Niranjan Awasthi, Jun Li, Margaret A. Schwarz, Urs von Holzen. Antitumor effect of cyclin- dependent kinase inhibitor flavopiridol in c-Myc overexpressing esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 975.

Effect of Linc-POU3F3 on radiotherapy resistance and cancer stem cell markers of esophageal cancer cells.

Long non-coding RNA (LncRNA) is an important transcriptional and post-transcriptional regulatory molecule in the body. In recent years, relationship between LncRNA and malignant phenotype of tumor cells has been revealed gradually. This study aims to investigate the expression characteristics of pit-oct-unc class 3 homeobox 3 related long non-coding RNA (Linc-POU3F3) in esophageal cancer and its relationship with radiation resistance (IR) as well as the expressions of cancer stem cell (CSC) markers in esophageal cancer cells. The expression characteristics and potential interaction molecules of Linc-POU3F3 in esophageal cancer were collected from the public database via bioinformatics retrieval. Forty-two pair samples of esophageal cancer tissues and corresponding adjacent tissues were collected. Human normal esophageal epithelial cells (HEEC) and human esophageal cancer cell lines (ECA109, TE-1, TE-2, TE-13) were cultured. Real-time quantitative PCR (qPCR) was used to detect the expression level of Linc-POU3F3 in clinical tissues and cells. The formation of TE-13 IR cell line induced by different doses of radiation served as IR group cells, and the same condition treated with 0 Gy dose was set as control group (control) cells. Meanwhile, we used cell transfection technology to construct random interference sequence (siControl) cells and interference (siLinc-POU3F3) cells. In ECA109 cells, we transfected blank and over expressed Linc-POU3F3 plasmids as vector and over-expressed group (oeLinc-POU3F3). The mRNA and protein expressions of CD44, CD133 and CD90 were detected by qPCR and Western blotting, respectively. MTS [3-(4,5-dimenthylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] was used to detect the cell viability under different radiation doses, and the resistance of IR cells was verified by clone formation experiment. The expression of Linc-POU3F3 was correlated with the tumor progression and poor prognosis of esophageal cancer. The level of Linc-POU3F3 mRNA expression was significantly higher in esophageal cancer tissues and cell lines than that in normal adjacent tissues and cell lines (all P<0.01). The expressions of Linc-POU3F3 mRNA and protein expressions of CD44, CD133, and CD90 in IR cells were significantly higher than those in control cells (all P<0.01). The expression of Linc-POU3F3 in siLinc-POU3F3 cell was significantly lower than that in the siControl cells (P<0.01), and the inhibition rate was 87.21%. The mRNA and protein expressions of CD44, CD133, and CD90 in the siLinc-POU3F3 cells were significantly lower than those in the siControl cells (all P<0.05). The expressions of linc-POU3F3, CD44, CD133, and CD90 mRNA and protein in the oeLinc-POU3F3 cells were significantly higher than those in the vector cells. The relative activity and clone formation ability in the IR cells were significantly higher than those in the control cells at 2, 4, and 8 Gy doses (all P<0.01). The relative activity in the siLinc-POU3F3 cells was significantly lower than that in the siControl cells at 4 and 8 Gy doses (P<0.01). The relative activity in the oeLinc-POU3F3 cells was significantly higher than that in the vector cells at 4 and 8 Gy doses (P<0.01). Linc-POU3F3 is up-regulated in esophageal cancer and can promote IR and the expression of CSC markers in esophageal cancer cells.

SERPINE2 promotes cellular migration and invasion in esophageal squamous cell carcinoma by activating β-catenin

Objective: To clarify the function and molecular mechanisms of serpin family E member 2 (SERPINE2) in cellular migration and invasion of esophageal squamous cell carcinoma (ESCC). Methods: The expression of SERPINE2 in ESCC was analyzed by using online databases TCGA (http: //gepia.cancer-pku.cn/detail.php and http: //ualcan.path.uab. edu/index.html). The expressions of SERPINE2 mRNA in normal human esophageal epithelial cell line NE2, human ESCC cell lines KYSE30 and KYSE150 were detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). SERPINE2-konckdown or SERPINE2-overexpressed plasmid was transfected into KYSE30 cells, and the efficiencies of the knockdown and overexpression system were tested by qRT-PCR. The relationships of SERPINE2 and ESCC migration and invasion were determined by migration and invasion assays in vitro. The associations between SERPINE2 expression and β-catenin as well as its target genes including c-Myc, cyclin D1 and CD44 were analyzed by immunofluorescence, qRT-PCR and western blot, respectively. Results: The expressions of SERPINE2 were significantly upregulated in both esophageal cancer (ESCA) and ESCC tissues compared to normal tissues by analyzing 182 and 95 cases, respectively (P<0.01). SERPINE2 is highly expressed in both KYSE30 and KYSE150 cells (P<0.05). The number of migrating and invading cells in control group were (212.66±24.11)/field and (136.00±14.42)/field, while were (88.33±9.71)/field and (77.00±9.53)/field in SERPINE2-knockdown 1 group, and (66.00±8.00)/field and (45.66±3.78)/field in SERPINE2-knockdown 2 group, respectively, and the differences were dramatically significant compared with the control group (P<0.01). The number of migrating and invading cells in control group were (250.00±30.00)/field and (203.33±15.27)/field, while were (383.33±35.11)/field and (246.66±25.16)/field in SERPINE2-overpressed group, and the differences were strikingly significant compared with the control group (P<0.01). The protein expression of β-catenin was upregulated while phosphorylated β-catenin protein expression was downregulated in SERPINE2-overexpressed KYSE30 cells when compared to control cells.The transcription activity of β-catenin was significantly upregulated and the mRNA expressions of its target genes including c-Myc, cyclin D1 and CD44 were all increased. After treated with 25 μM iCRT14, the number of migrated cells in the control and SERPINE2-overpressed groups were (200.00±36.05)/field and (258.33±22.54)/field, and the number of invaded cells were (160.00±17.32)/field and (188.33±25.65)/field, respectively, the differences were dramatically significant compared with the group without iCRT14 treatment (P<0.01). Conclusion: SERPINE2 is significantly upregulated in ESCC cells and can promote cellular migration and invasion by activating β-catenin, which may provide a potential therapeutic target for patients with ESCC.

High expression of guanine nucleotide-binding protein-like-3-like is associated with poor prognosis in esophageal cancer.

Guanine nucleotide-binding protein-like-3-like (GNL3L) is required for processing ribosomal pre-rRNA and cell proliferation and is upregulated in many types of cancer. This study is aimed to investigate the clinical significance of GNL3L in esophageal cancer. The mRNA and protein expression levels of GNL3L were determined by using quantitative real-time polymerase chain reaction and immunohistochemistry, respectively. GNL3L was localized in both cytoplasm and nucleus. The expression levels of GNL3L in esophageal cancer tissues were significantly higher than those in adjacent nonmalignant tissues. High GNL3L expression was associated with pathologic type and poor differentiation. Patients with high GNL3L expression had shorter overall survival (OS) than those with low GNL3L expression. Multivariate Cox regression analysis revealed that GNL3L expression was an independently predictive factor for the OS of patient with esophageal cancer. The Gene Expression Profiling Interactive Analysis (GEPIA) databases also showed that GNL3L was upregulated in esophageal cancer, which was closely associated with an unfavorable prognosis of patients with esophageal cancer. Taken together, our findings suggest that GNL3L is upregulated in esophageal cancer, which is linked to the progression of the disease. As a result, GNL3L could be used as a biomarker for esophageal cancer.

CircRNA VIM silence synergizes with sevoflurane to inhibit immune escape and multiple oncogenic activities of esophageal cancer by simultaneously regulating miR-124/PD-L1 axis.

Circular RNA of vimentin (circ-VIM) is a predictor for poor prognosis of acute myeloid leukemia, but we had little information on its function in esophageal cancer (EC). Here we examined the effects of circ-VIM together with sevoflurane on immune escape and multiple oncogenic activities of EC. Bioinformatic tools, luciferase assay, and RNA immunoprecipitation were used to examine regulations between circ-VIM, miR-124-3p (miR-124), and PD-L1. CCK-8, wound healing, and Transwell assays were used to measure cell proliferation, migration, and invasion, respectively. The impacts of EC cells on cytotoxicity, proliferation, and apoptosis of CD8+ T cells were examined using LDH assay, CFSE staining, and Annexin V/PI staining, respectively. The in vivo tumorigenesis and lung metastases were assessed using xenograft model and tail vein injection of EC cells. Significant upregulation of circ-VIM and PD-L1 and downregulation of miR-124 were detected in EC tissues or cells. Circ-VIM sponged miR-124 and released its suppression on the downstream target PD-L1. Sevoflurane, independent of circ-VIM, also upregulated miR-124 to lower PD-L1 expression. By modulating miR-124/PD-L1 axis, silencing circ-VIM and applying sevoflurane both inhibited immune escape and multiple oncogenic activities of EC in vitro, and suppressed xenograft growth and lung metastases in vivo. The inactivation of Ras/ERK signaling pathway was involved in suppression of malignant phenotypes by silencing circ-VIM and sevoflurane treatment. Silencing circ-VIM and applying sevoflurane, by separately regulating miR-124/PD-L1 axis, presented synergistic effects in inhibiting immune escape and multiple malignant phenotypes of EC cells.